The pilus of Neisseria gonorrhoeae is a major virulence factor that mediates essential adherence functions for the bacterium. The only known protein contained in the pilus is pilin. This protein shows great antigenic diversity in different strains, and also undergoes a high rate of antigenic variation within individual strains. Pilin antigenic variation is a major reason that immunity to gonococcal reinfection has never been documented, even in repeatedly infected populations. The pilin amino acid changes that lead to antigenic variation within laboratory strains and a limited number of clinical isolates have been extensively investigated. However, large scale studies examining the diversity of pilus antigenic variation in clinical isolates has not been reported. Previous studies on pilus serology have searched for common pilus epitopes in order to design cross reactive subunit vaccines or to identify specific targets for diagnostic reagents; it was concluded that there were no cross reactiveepitopes that could be used for these purposes. At the time most of these studies were conducted the molecular basis for pilin antigenic variation had not been determined, and correlations between antibody cross reactivity and amino acid sequence were not addressed. We propose to screen a panel of fresh clinical strains with matched patient sera in order to characterize how much pilus antigenic variation occurs in vivo. We will also determine the predicted pilin amino acid sequences of some of these strains to characterize if different amino acid changes occur in natural infections than in laboratory culture. We will collect matched sets of strains, sera, and genital swabs from patients who have never had gonorrhea before, and use nonpiliated phase variants derived from each isolate to absorb out antibodies that react with non-pilus surface components of each bacterium. The absorbed "pilus-specific" sera will be used in a solid phase fluorescence immunoassay to test the anti-pilus titer of each sera against each piliated isolate. This will allow antigenically related pili to be grouped with respect to human sera reactivity. The sera will then be used to screen larger numbers of clinical isolates for those that express pili that fall into these serogroups. From this analysis, the diversity of pilus antigenic variants in vivo will be determined. The predicted amino acid sequence of selected members of each serogroup will be determined by PCR amplification of the expressed pilin gene and DNA sequence analysis. Thus the primary pilin protein sequence will be compared to antibody cross reactivity to determine if a multivalent, pilus based vaccine that contains highly cross reactive pilus proteins from every serogroup could be designed, which would protect against bacteria expressing most natural pilus types. A second major goal is to determine the primary amino acid sequence of natural pilin variants, compare the sequences in those expressed in the laboratory, and attempt to correlate those sequences with antibody reactivity.